Coenzyme B12-dependent processes are vital to human health and play important roles in ecological niches and industrial applications. The long-term goal of the proposed research is to improve our general understanding of the genetics and biochemistry of coenzyme B 12. The proposed studies focus on the metabolism of inactive cobalamins into the B 12 coenzymes. This process is needed for the assimilation of exogenous cobalamins, and because the B 12 coenzymes are unstable, for the maintenance of adequate B 12 pools to meet physiological needs. The human cob(I)alamin adenosyltransferase which functions in cobalamin metabolism will be characterized biochemically and its 3-dimensional structure will be determined. The reduction of cob(II) alamin to cob(I)alamin for coenzyme B 12 synthesis will also be investigated. Preliminary studies showed that the human methionine synthase reductase can reduce cob(II)alamin to cob(I)alamin for coenzyme B 12 synthesis, and results indicated a highly specific interaction between the human methionine synthase reductase and adenosyltransferase enzymes. The reduction of cob(II)alamin to cob(I)alamin by the human methionine synthase reductase will be more fully characterized. The results of bioinformatic analyses have indicated that the bacterial YgfD protein and its homologues are involved in cobalamin metabolism The specific function of the YgfD protein will be determined by a series of biochemical tests. The final aim of the project will be to use the genes identified in this study to construct gene therapy vectors for expression of enzymes involved in cobalamin metabolism. The proposed studies will provide information directly applicable to improved methods of diagnosis and treatment of inherited diseases of B 12 metabolism. Furthermore, since changes in B 12 metabolism have been linked to chronic liver disease, cancer, elevated serum homocysteine (a possible risk factor in heart disease), increased rates of DNA damage, impaired cognitive function, and HIV infection, the proposed studies may also provide information relevant to understanding these diseases.